Skip to main content

Combined effects of an antioxidant and caspase inhibitor on the reversal of hepatic fibrosis in rats

Abstract

We sought to determine the hepatic fibrosis-reversal effects upon simultaneous administration of lithospermate B (LAB), an anti-oxidant, and nivocasan, a caspase inhibitor, to rats compared with each compound alone. Liver fibrosis was induced in Sprague–Dawley rats by thioacetamide (TAA). Rats were treated with TAA and then given LAB and (or) nivocasan. Fibrotic areas were evaluated quantitatively by computerized morphometry. Apoptosis was assessed using a TUNEL assay, and immunohistochemical staining for malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4HNE) was performed to assess oxidative stress levels. Real-time quantitative PCR was used to quantify expression of fibrosis-related genes. The degree of hepatic fibrosis was significantly reduced in rats treated with LAB and nivocasan compared to either treatment alone (P < 0.001). Treatment with each compound significantly decreased expression of fibrosis-related genes, such as type I collagen α1 (col1α1), α-SMA and TGF-β1 (P < 0.05). Co-treatment with LAB and nivocasan further reduced col1α1 expression compared to treatment with either compound. A TUNEL assay revealed that hepatocyte apoptosis was significantly decreased in the group treated with nivocasan compared to other groups (P < 0.01). Immunohistochemistry showed a decrease in MDA and 4HNE, reflecting amelioration of oxidative stress, when LAB or LAB+nivocasan was administered compared to nivocasan alone (P < 0.01). Nivocasan was found to inhibit caspase-1, -3, -7, -9 and gliotoxin-induced death of rat-derived hepatic stellate cells was inhibited by nivocasan administration without overexpression of α-SMA. Conclusions: Co-incidental administration of LAB and nivocasan suppressed oxidative stress and apoptosis, resulting in enhanced reversal of hepatic fibrosis in rat.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

References

  1. Friedman SL (2008) Mechanisms of hepatic fibrogenesis. Gastroenterology 134:1655–1669

    PubMed  Article  CAS  Google Scholar 

  2. Bataller R, Brenner DA (2005) Liver fibrosis. J Clin Invest 115:209–218

    PubMed  CAS  Google Scholar 

  3. Ellis RE, Yuan JY, Horvitz HR (1991) Mechanisms and functions of cell death. Annu Rev Cell Biol 7:663–698

    PubMed  Article  CAS  Google Scholar 

  4. Reed JC, Tomaselli KJ (2000) Drug discovery opportunities from apoptosis research. Curr Opin Biotechnol 11:586–592

    PubMed  Article  CAS  Google Scholar 

  5. Nicholson DW (2000) From bench to clinic with apoptosis-based therapeutic agents. Nature 407:810–816

    PubMed  Article  CAS  Google Scholar 

  6. Reed JC (2002) Apoptosis-based therapies. Nat Rev Drug Discov 1:111–121

    PubMed  Article  CAS  Google Scholar 

  7. MacBride CB, McPhail LT, Steeves JD (1999) Emerging therapeutic targets in caspase-dependent disease. Emerg Ther Targets 3:391–411

    Article  Google Scholar 

  8. Patel T (2000) Apoptosis in hepatic pathophysiology. Clin Liver Dis 4:295–317

    PubMed  Article  CAS  Google Scholar 

  9. Thorburn A (2004) Death receptor-induced cell killing. Cell Signal 16:139–144

    PubMed  Article  CAS  Google Scholar 

  10. Parola M, Robino G (2001) Oxidative stress-related molecules and liver fibrosis. J Hepatol 35:297–306

    PubMed  Article  CAS  Google Scholar 

  11. Koek GH, Liedorp PR, Bast A (2011) The role of oxidative stress in non-alcoholic steatohepatitis. Clin Chim Acta 412:1297–1305

    PubMed  Article  CAS  Google Scholar 

  12. Nieto N, Greenwel P, Friedman SL, Zhang F, Dannenberg AJ, Cederbaum AI (2000) Ethanol and arachidonic acid increase alpha 2(I) collagen expression in rat hepatic stellate cells overexpressing cytochrome P450 2EI. Role of H2O2 and cyclooxygenase-2. J Bio Chem 275:20136–20145

    Article  CAS  Google Scholar 

  13. Kim KM, Kim YM, Park M, Park K, Chang HK, Park TK et al (2000) A broad-spectrum caspase inhibitor blocks concanavalin A-induced hepatitis in mice. Clin Immunol 97:221–233

    PubMed  Article  CAS  Google Scholar 

  14. Paik YH, Yoon YJ, Lee HC, Jung MK, Kang SH, Chung SI et al (2011) Antifibrotic effects of magnesium lithospermate B on hepatic stellate cells and thioacetamide-induced cirrhotic rats. Exp Mol Med 43:341–349

    PubMed  Article  CAS  Google Scholar 

  15. Jung M, Lee HC, Ahn CW, Park W, Choi S, Kim H et al (2002) Effective isolation of magnesium lithospermate B and its inhibition of aldose reductase and fibronectin on mesangial cell line. Chem Pharm Bull 50:1135–1136

    PubMed  Article  CAS  Google Scholar 

  16. Malekzadeh R, Mohamadnejad M, Rakhshani N, Nasseri-Moghaddam S, Merat S, Tavangar SM et al (2004) Reversibility of cirrhosis in chronic hepatitis B. Clin Gastroenterol Hepatol 2:344–347

    PubMed  Article  Google Scholar 

  17. Pockros PJ (2009) Antifibrotics for chronic hepatitis C. Clin Liver Dis 13:365–373

    PubMed  Article  Google Scholar 

  18. Liaw YF (2011) Impact of hepatitis B therapy on the long-term outcome of liver disease. Liver Int 31(Suppl 1):117–121

    PubMed  Article  Google Scholar 

  19. Guicciardi ME, Gores GJ (2005) Apoptosis: a mechanism of acute and chronic liver injury. Gut 54:1024–1033

    PubMed  Article  CAS  Google Scholar 

  20. Anstee QM, Concas D, Kudo H, Levene A, Pollard J, Charlton P et al (2010) Impact of pan-caspase inhibition in animal models of established steatosis and non-alcoholic steatohepatitis. J Hepatol 53:542–550

    PubMed  Article  CAS  Google Scholar 

  21. Pockros PJ, Schiff ER, Shiffman ML, McHutchison JG, Gish RG, Afdhal NH et al (2007) Oral IDN-6556, an antiapoptotic caspase inhibitor, may lower aminotransferase activity in patients with chronic hepatitis C. Hepatology 46:324–329

    PubMed  Article  CAS  Google Scholar 

  22. DeMinicis S, Seki E, Oesterreicher C, Schnable B, Schwabe RF, Brenner DA (2008) Reduced nicotinamide adenine dinucleotide phosphate oxidase mediates fibrotic and inflammatory effects of leptin on hepatic stellate cell (HSC). Hepatology 48:2016–2026

    Article  CAS  Google Scholar 

  23. Ratziu V, Sheikh MY, Sanyal AJ, Lim JK, Conjeevaram H, Chalasani N et al (2012) A phase 2, randomized, double-blind, placebo-controlled study of GS-9450 in subjects with nonalcoholic steatohepatitis. Hepatology 55:419–428

    PubMed  Article  CAS  Google Scholar 

  24. Hsu YC, Lin YL, Chiu YT, Shiao MS, Lee CY, Huang YT (2005) Antifibrotic effects of Salvia miltiorrhiza on dimethylnitrosamine-intoxicated rats. J Biomed Sci 12:185–195

    PubMed  Article  CAS  Google Scholar 

  25. Canbay A, Higuchi H, Bronk SF, Taniai M, Sebo TJ, Gores GJ (2002) Fas enhances fibrogenesis in the bile duct ligated mouse: a link between apoptosis and fibrosis. Gastroenterology 123:1323–1330

    PubMed  Article  CAS  Google Scholar 

  26. Song E, Lee SK, Wang J, Ince N, Ouyang N, Min J et al (2003) RNA interference targeting Fas protects mice from fulminant hepatitis. Nat Med 9:347–351

    PubMed  Article  CAS  Google Scholar 

  27. Canbay A, Feldstein A, Baskin-Bey E, Bronk SF, Gores GJ (2004) The caspase inhibitor IDN-6556 attenuates hepatic injury and fibrosis in the bile duct ligated mouse. J Pharmacol Exp Ther 308:1191–1196

    PubMed  Article  CAS  Google Scholar 

  28. Strobel Swanson L, Korsmeyer S, Cannistra SA (1996) BAX enhances paclitaxel-induced apoptosis through a p53-independent pathway. Proc Natl Acad Sci USA 93:14094–14099

    PubMed  Article  CAS  Google Scholar 

  29. Masuoka HC, Guicciardi ME, Gores GJ (2009) Caspase inhibitors for the treatment of hepatitis C. Clin Liver Dis 13:467–475

    PubMed  Article  Google Scholar 

  30. Nakamoto Y, Kaneko S, Fan H, Momoi T, Tsutsui H, Nakanishi K et al (2002) Prevention of hepatocellular carcinoma development associated with chronic hepatitis B by anti-fas ligand antibody therapy. J Exp Med 196:1105–1111

    PubMed  Article  CAS  Google Scholar 

  31. MacDonald GA, Bridle KR, Ward PJ, Walker NI, Houglum K, George DK et al (2001) Lipid peroxidation in hepatic steatosis in humans is associated with hepatic fibrosis and occurs predominantly in acinar zone 3. J Gastroenterol Hepatol 16:599–606

    PubMed  Article  CAS  Google Scholar 

  32. Zhan SS, Jiang JX, Wu J, Halsted C, Friedman SL, Zern MA et al (2006) Phagocytosis of apoptotic bodies by hepatic stellate cells induces NADPH oxidase and is associated with liver fibrosis in vivo. Hepatology 43:435–443

    PubMed  Article  CAS  Google Scholar 

  33. Jiang JX, Mikami K, Venugopal S, Li Y, Torok NJ (2009) Apoptotic body engulfment by hepatic stellate cells promotes their survival by the JAK/STAT and Akt/NF-kappaB-dependent pathways. J Hepatol 51:139–148

    PubMed  Article  CAS  Google Scholar 

  34. Nieto N, Greenwel P, Friedman SL, Zhang F, Dannenberg AJ, Cederbaum AI (2000) Ethanol and arachidonic acid increase alpha 2(I) collagen expression in rat hepatic stellate cells overexpressing cytochrome P450 2E1. Role of H2O2 and cyclooxygenase-2. J Biol Chem 275:20136–20145

    PubMed  Article  CAS  Google Scholar 

  35. Gu K, Zhao JD, Ren ZG, Ma NY, Lai ST, Wang J et al (2011) A natural process of cirrhosis resolution and deceleration of liver regeneration after thioacetamide withdrawal in a rat model. Mol Biol Rep 38:1687–1696

    PubMed  Article  CAS  Google Scholar 

Download references

Acknowledgments

This study was funded in part by a Korea Healthcare Technology R&D Project from the Ministry for Health and Welfare, Republic of Korea (HI10C2020), and supported in part by a faculty research grant of Yonsei University College of Medicine for 2010 (6-2010-0032).

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Kwang-Hyub Han.

Additional information

Do Young Kim and Sook In Chung contributed equally to this work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 11 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Kim, D.Y., Chung, S.I., Ro, S.W. et al. Combined effects of an antioxidant and caspase inhibitor on the reversal of hepatic fibrosis in rats. Apoptosis 18, 1481–1491 (2013). https://doi.org/10.1007/s10495-013-0896-5

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10495-013-0896-5

Keywords

  • Hepatic fibrosis
  • Caspase inhibitor
  • Antioxidant
  • Rat